Stock No: 000453
Protocol 13902: Pyrosequencing Assay - Ank1<nb>
Version 2.0

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Sequence

TCTTTGCCTACAGAGCTGGCCCGG[g]AGCTGCAGTTCAGCGTGGAAGACATC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10794 GGC TCT TGG TTC TTT GCC TAC A Forward
10795 Fluorophore GTC TTC CAC GCT GAA CTG CA Reverse
10796 ACA GAG CTG GCC CGG SEQ

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
10794 0.50
0.50
Glycerol 5.00
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
000448 B6.Cg-Ank1nb/BrkJ
000453 WB.Cg-Ank1nb/BrkJ
2 strains use this protocol